Method for preparing folic acid intermediates



Patented Aug. 12, 1952 UNITED STATES PATENT OFFICE METHODFOR PREPARING FOLIC ACID INTERMEDIATES Barney J. Magerlein and David I. Weisblat, Kala-. maz'oo, Mich., assignors to The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan No Drawing. Application August 19,1950, Serial No. 180,502

4 N 6 CH -N\-CO(NECHCHiCH2OO)nOR HN I2 7 R arylSOg \NAN/ N-( (2-amino-ei-hydroxy-G-pteridyl)-methy1)-p-aminobeuzoate compound wherein R is a member of the-class consis ting of hydrogen and the alkyl radicals, 11 is a-member of the class consisting of zero and the pos itive integers 1 to '7, inclusive, and R. is a member of the class consisting of hydrogen and the alkyl radicals. They thus include the arylsulfonyl derivatives of pteroic acid, of pteroylglutamic acid and ,of the pteroylglutamyl glutamic acids having up to seven glutamic acid residues in the molecule, and of their alkyl esters as well as alkyl substitution products thereof having an alkyl substituent on. the 7 carbon atom of the pterine nucleus. In the structural formulae given herein and in the appended claims aromatic nuclei are represented by one or more simple hexagons.

In the naming of compounds having the generic formula given, certain of which are described and claimed in copending application, Serial No. 41,882, filed July 31, 1948, now Patent No. 2,558,711, and of other starting and intermediate compounds mentioned herein where both a glutamic acid residue and a p-amino-benzoic acid residue are included in the molecule, the nitrogen atom of the glutamic acid residue is. for convenience, herein referred to by the symbol N and the nitrogen atom of the p-aminobenzoic acid residue is referred to by the symbol N. As indicated by the generic formula given, compounds used in or prepared by the process of the invention which contain more than one stood, however, that the invention is not limited V thereby.

8 Claims. (Cl. 260251.5)

Compounds having the generic formula (I) and compoundsuseful as intermediates in the preparation thereof, are of particular value due to the relationship of certain of the former with;

and to the ease with which they can be converted to, compounds similar to or identical with certain naturally occurring compounds of the folic acid group.- Thus. diethyl N-(N-((2- amino 4-hydroxy-6-pteridyl) -methyl) -,N (p- 'toluene sulfonyl) p aminobenzoyl) 1- glutamate, which can be prepared by reacting 2,4,5-triamino-6-hydroxyprimidine with diethyl N (N (2- keto 3 oximinopropyl) N-' (p-toluenesulfonyl) -p-aminobenzoyl) -glutamate according to the method of the invention, can be converted readily by hydrolysis of the two ethyl ester groups with alkali to the corresponding dicarboxylic acid compound and the latter, by splitting the p-toluenesulfonyl radical from the molecule with hydrogen bromide and a'bromine' acceptor in an aliphatic acid medium according to the method described and claimed in copending application, Serial No. 41,883, filed July 31, 1948, now Patent No. 2,562,222, can be converted readily to a pteroyl-glutamic acid which is generally accepted as being identical with vitamin B0 from liver or the L. casei factor. The order of the hydrolysis step and the splitting of the arylsulfonyl radical can be reversed, if desired- Compounds having the generic formula given in which th gl acid residues have the 1 configuration are of particular interest because of the existence of glutamic acid residues having the same configuration in folic acid compounds isolated from natural sources. the invention and the intermediate compounds to be described are, however, not limited as to the configuration of the glutamic acid residues involved. i

c 0 OR R-cm-o 0CH2N-C -C omno'rromomo 0),.012'

' aryl O1 0 V N-(2-ketoalkyl)-p aminobenzoate compound l RONO R-d-o o-cHz-N-O-oomrrhrromomo O)OR aryl Oz N-(2-ketc-3-cximinoalkyD-p-aminobenzoate compound 2,4,5-triamlno-6-hydroxypyrimidine The method of 1 COOR" 7 aryl SO;

N-((2-amino-4-hydroxy-6-pteridyl)-inethyl)-aminobenzoa te compound latter compound-can .be-prepared readily by re acting an alkyl nitrite in either an acid or an alkaline medium, preferably in an acid medium,-

with an N -(2-keto-alkyl) -paminobenzoate compound having the formula. (II).

It is-to be noted that when the 2-keto-3-ox iminoalkyl radical of the N-(2-keto-3-oximinoalkyD-p-aminobenzoate compound v(III) contains more. than three carbon atoms,- 1. e. when Roi the formulae given is an alkyl radical, the N ((2 amino 4 hydroxy 6 pteridyl) methyl).-p-aminobenzoate compound (I) formed is an N4(Z-amino-d-hydroxyfl-all:yl-6-pteri dyl) 'nethyl) -.p-aminobenzoate compound, there being an'alkyl substituent in. the '7 position of the. pterine nucleus.

dissolving the compounds in an organic liquid containing either an acid or an alkaline catalyst.

A variety of organic liquids can be employed, such as dioxane, diethyl ether, alcohol, tetra hydrofurane and the like. An organic liquid is preferably chosen in which the acid or alkaline catalyst is at least somewhat soluble. Ethereal solutions of hydrogen chloride and alcoholic solutions of alkali metal alcoholates have been used with satisfaction. Other acids and other alkaline substances can, however, be used, if desired.

Alkyl nitrites which can be used include n-butyl nitrite, tertiary-butyl nitrite, iso-octyl nitrite, ethyl nitrite and the like. Substantially one mole, or somewhat more, of an alkyl nitrite is used for each mole of 'ketoalkyl compound. The reaction mixture is generally allowed to stand at room temperature for from 6 to 12 hours, but higher temperatures can be employed if desired and the reaction time shortened accordingly.

When an acid catalyst is used to promote the reaction of an alkyl nitrite with the ketoalkyl compound, the keto-oximinoalkyl compound can be isolated conveniently by evaporating the volatile components of the mixture under reduced cng-a oc oomcncnicnlc o ..o n I Such N((2-aminolhy-' dTOXY alkyl merino methyl) pressure' When the reaction is carried out in an alkaline medium, the keto-oximinoalkyl compound can be isolated readily by washing the reaction mixture with aqueous acid, subsequently the washed water-insoluble portion and evaporating under reduced pressure any volatile components which may be present. The N-(2- keto 3 oximinoalkyl) p aminobenzoate compound is thus generally recovered as a viscous yellow sirup which is sufiiciently pure for most purposes.-

The reaction of 2,4,5-triamino-6-hydroxypyrimidine with an N-(2-keto-3-oximinoalky1)-pvaminobenzoate compound is carried out readily by mixing the substances together, e. g., in glacial acetic acid, preferably with the addition of anhydrous sodium acetate, and allowing the mixture to stand, e. g. at room temperaturefor about 30 minutes, and then heating under reflux, e. g.,

for about 2 hours or longer. The 2,4,5-triamino- G-hydroxypyrimidine can be used in the form of its dihydrochloridefor greater ease in handling, if desired. The reaction is preferably carried out under an atmosphere or" nitrogen or other inert gas to prevent the formation of undesirable oxidation products. The reaction conditions can be varied considerably from those mentioned and such variations are contemplated by the invention.

Following the heating step the reaction mixture can be worked up by distilling the acetic acid under reduced pressure and washing the residue with water to remove inorganic salts and anyremaining acetic acid. The N-( (2-amino-4- hydroxy pteridyl) methyl) p aminobenzoate compound is thus obtained as a highly colored residue. The compound thus'obtained can be converted directly and Without further purification by hydrolysis, in the case of esters,

and by cleavage of the arylsulfonyl radical from the molecule, to products containing a substantial proportion of pteroic acid or a pteroyl glutamic acid, depending upon the absence or presence of glutamic acid residues in the keto-oximinoalkyl compound.

An 'N- (Z-ketoalkyl) -p-amincbenzoate compound (II) useful as a starting compound in the method of the present invention can be prepared as described and claimed in concurrently filed copending application, Serial No. 180,501, by first reacting a 1,2-epoxyalkane (V) having the formula 1,2epoxyalkane with an N-(arylsulfonyl) -p-aminobenzoate estercompound (VI) having the formida r l J QOOalkyl HN .c munch- 011201120 0),.0 allryl aryl O;

v i VI 'VN- (arylsulfonyl) -paminobenzoate ester compound to form an g? (Z-hydroXyalkyI)'-p-aminobenzoate ester compound (VII) having the formula.

7 COOallryl.

R'CH -CHOH+C-H -NC c 0 (NE on culorco o .0 allryl' aryl S02 .VII'. N-( 2-liydroxyalk.yl)paininobenzoate ester compound and subsequently oxidizing the hydroxyalkyl com pound, e. g. with chromic anhydride in glacial acetic acid. Hydrolysis to the.=free acidcan;be

effected :either before jor:.aft,er oxidation. alternatev method by which the oxidation canbe carried out using an aqueous acid-solutionof a dichromateflisudescribedand claimed in concur:

rently filed copending application, Serial; No

180,507.; N'-(Arylsulfonyl)ep-aminobenzoate jester compounds (VI) .which can be employed with theproduction of the corresponding intermediate and finalcompounds ofthe process include the alkyl- N ,(arylsulfonyl) -p-amino-benzoates, the dialkyl N"-.,N.-l(arylsulfonyl) -,p-aminobenzoyl)- glutamates,,.the alkyl' Nf-(Nearylsulfonyh-paminobenzoyl)-glutamyl-glutamates having up to. 7. glutamic acid residues in the molecule and the corresponding free acids.

a 'Certain :of the N- (arylsulfonyl) -p-aminoben'- zoate compoundsare described and .claimed in CO? pending application-Serial No. 41*,888yfi-1ed July- 31, 1948. They can be preparedreadily as .de-

scribedtherein by reacting an arylsulfonyl halide with, p-aminobeh'zoic acid, N (p-aminobenzoyD glutamic acid, or an N'-(p-aminobenz'oyl)'-g1utamyl-glutarnic acid having up to '7 glutamic acid residues in the molecule or with'an, alkyl ester thereon? .N (Ar-ylsulfonyl) p s aminobenzoate; compounds which are esters can beh-ydrolyzed usediin the process, although insofarasis ,known';

. .40; Although'starting compounds containing sub-.

any alkyl ester-can be used.

stantially any arylsulfonyl radical can be used in the process of the invention and in the prepara-,.

tion of the corresponding arylsulfonyl intermediates, and .final products, the preferred starting materialsand intermediate compounds are those containing the p-toluenesulfonyl radical due to the-ready availability of the p-toluenesulfonyl halides and to the generally crystallinenature of the p-toluenesulfonyl derivatives of compounds withwhichgthe present invention is concerned. Furthermore, it appears that the p-toluensulfonyl radical can frequently be, removed from the compounds involvedherein e. g. by splitting with hydrogen bromideas mentioned previously. somewhat more readily and with the formation of a smaller proportion of .undesirable by-products than in the case of some of the other arylsulfonyl radicals. The invention is, however, not limited to; eompounds containing the p-toluenesulfonyl radical and compounds containing other arylsulfon'yl; radicals, such .flas the o-toluenesulfonyl,

radicals'j'and the like,fca'n beused if desired. f It" should also be mentioned that compounds containing arylsulfonyl radicals having non-hydrocarhon substituents can be used in'the process andthe corresponding intermediate and ,final naphthalenesulfonyl, methylnaphthalenesulfonyl compoundsprepared. provided only that the substituent isnon-reactiveunder the reaction conditi'ons v Such non-reactive substituents include chlorine, bromine, and-the-methoxy, phenoxy, nitro and other radicals 1 ;-A lthough R is the-formulae givenrepresents hydrogen or anyalk-yl radical the preferred com--.

in conventional mane.

pounds are those wherein'R is hydrogen and the invention has been described with particular ref- Among the alkyl radicals that,

erence thereto. can. be used. in the process, if desired, are the methyl, ethyl, n-propyl, iso-pro'pyl, n-butyl, cert.- butyl and octyl radicals as well as many others.

Certain. advantages. of the invention are apparentfrom the following examples which, are

iven yway of illustration only and are not to be construed as limiting.

Example 1Q-Diethyl N'-(N-(p-ioluenesulfonylip-ammobenzoyl) -glutamaie Thirty andnine-tenths grams of D-tOIuenesuI fonyl-p-aminobenzoyl chloride and 23.9 grams of diethyl 1,(+)-g1utamate hydrochloride were dissolved in 300 milliliters of ethylene dichloride and the solution cooled to between 0 and 10 C. The 'cold solution was stirred vigorously and-22.3 grams of'triethylamine in 72 milliliters of ethylene dichloride was added slowly over a periodof about 20 minutes. The temperature of the mixture was held between 10 and 20C. during the addition of the triethylamine and'the tolstand at room temperature for one hour, The mixture was then washed successivelywith'water, dilute hydrochloric acid, saturated aqueous sodium bicarbonate and finally with water. The' colorless solution thus obtained was dried with anhydrous sodium sulfate and naphtha was added until the solution became opalescent. The mixture was then cooled to cause crystallization and filtered; The crystals, after drying, consisted of 36 grams of diethyl N'-(N-(p-toluenesulfonyl)- p-aminobenzoyl) -l-glutamate melting at 124 to 126 C. i

Emma ,--2 Dieth yl N-(N- lad-rowan Nr (ptoluenesulfonyl) -p e aminobenzoyli-glue .ita'mate A .7

jA mixture of 6.3 8 gramslof V 47.7 grams of diethyl N -(N ip-toluenesulfonyl) p=aminobenzoyl7glutamate and 10 drops of an hydrous pyridine was heated under pressureat 130 C. for '2 hours. The reaction mixture was then cooled to room temperature, 400 milliliters of benzene were added'and the resulting benzene solution was washed with 50 milliliters of water and 50 milliliters of dilute hydrochloric acid and then dried. Removal of the solvent underreduced pressure gave a non-crystalline solid resi-j duce of diethyl N'-(N- (2-hydroxypropyl) -N-(p toluenesulfonyl) -p-aminobenzoyl) -glutamate.

In a similar manner, and using 1,2-epoxy-n-' butane or l,2'-epoxy-4-methyl-n-pentane in place of 1,2-ep0xypropane, there are formed diethyl N (N 2 -hydroxy-n-butyl) -N- (p-toluenesulfon yl) -p-aminobenzoyl) -glutamate and diethyl N'- (N-(2-hydroxy 4 methyl n-penty1) N (p-tolu-f enesulfonyl) p aminobenzoyD glutamate, respectively", which, upon hydrolysis, give the corresponding acids.

The useof ethylN (benzenesulriiyn mi-also enzen 9 t i i 'f- (N pe' be nesulfonyl) -p-aminobenzoyl) -g l utam'yl glutamate aminobe nzoyl): glutamate leads, in analogous;-

glutamate, respectively. These esters, upon hy drolysis with dilute alcoholic sodium hydroxide, J

are 1 converted to N -(2-hydroxypropyl) -N- '(benzenesulfonyl)-p-aminobenzoic acid and 'N'-(N mixture then allowed "propylene oxide,

1n place of diethyl N"- (N- (p toluenesulfonyD p- I (2 -hydroxypropyl) N p-chlorobenzenesulfonyl) -p-aminobenzoyl) -glutamyl-glutamic acid, respectively. The free acids mentioned, upon esterification with an alkanol or with other-alkyl esterforming agents, are converted to the corresponding alkyl esters.

Upon treating the Z-hydroxyalkyl compounds mentioned which are arylsulionyl :derivatives with hydrogen bromide according to the procedure described in the copending application previously referred to, the arylsulfonyl radical is split from the molecule leaving the corresponding Z-hydroxyalkylamino compound. The latter, upon reaction with an arylsulionyl halide in substantially conventional fashion, are converted readily to the corresponding arylsulfonyl derivatives or the amine.

Example s-rbzemyz N'-(N-(2 ketopropy-l) N- (p toluenesuljonyll .p amino-benzoyl) glutamate The crude diethyl N'-('N(2-hydroxypropyl) N"- (p-toluenesulfonyl) -p-aminobenzoyl). -glutamate obtained in Example 2 was dissolved. in 400 milliliters of benzene and the solution was added with vigorous stirring to a solution consisting of 52.6 grams of potassium dichromate, 230 milliliters of water, 33 milliliters of acetic acidand 69 milliliters of sulfuric acid; The mixture was stirred vigorously at to C. for about 2 /2 hours, cooled and the benzene and aqueous layers separated. The aqueous layer was extractedtwice with lilo-milliliter portions of benzene and then discarded, the benzene extracts being added to the benzene layer from the reaction mixture. The combined benzene solutions were washed three times with Z-milliliter portions of water, once with milliliters of saturated aqueous sodium bicarbonate solution, twice with 250-milliliter portions of water and oncev with 100 -milliliters of saturated aqueous sodium chloride solution. The washed benzene solution was then dried and the solvent distilled under reduced pressure. The residue was dissolved in hot iso-propanol and the solution 2.1.

lowed to crystallize at room temperature. The mixture was filtered and the crystals dried. There were thus. obtained 34.5 grams of crude diethyl N-(N-(2-ketopropyl) -N(p tcluenesulfonyl) p aminobenzoyl) glutamate meltedat 88 to C'. Recrystallization of the crude ketone raisedv its melting point to 99 to In an. alternate, procedure, diethyl. N-(N-(2- hydroxypropyl).-N-(p-toluenesulfonyl)' pami- 1 nobenzoyD-glutamate and a'small excess over the theoretical amount. of chro'mic anhydride are dissolved in glacial acetic acid'and the mixture allowed to stand'ior several'hours with occasional agitation at about 5 'to' 10 C. The mix ture is then stirred with ether and filtered. The ethereal solution is washed with water and aque ous bicarbonate, dried and the ether evaporated under reduced pressure. Diethyl N'-(N-(2-ketopropyl) -N (p toluenesulfonyl) -p-aminoben-.,

ZQyD-glutamate is thus obtained as a yellowish viscous residue which can be purified as previously described.

Oxidation in a similar manner of diethyl N- (N-(2-hydroxy-n-butyl) N (p toluenesulfonyl) p-aminobenzoyl) -glutamate, diethyl N'-(N (2 v hydroxy-4-methyln-pentyl) -N-(p-toluene sulfonyl) -p aminobenzoyl) glutamate, N'-(N (2-hydroxy-n-butyl) -N (p-toluenesulfonyl) -paminobenzoyl) -glutamic acid, N-(2-hydroxyprowhich 8 pyl) -N- (b'enzenesulfonyl) -'p-aminobenzoic acid, ethyl N -(2t-hydroxypropyl) N- (benzenesulfonyl)'-p-'aminobenzoate, triethyl N."-(N-(2 -hydro- Xypropyl) N-.(p-chlorobenzenesulfonyl)'-p-aminobenzoyl) glutamyl-glutamate and N'-(N-(2- hydroxypropyl) N-(p-chlorotoluenesulfonyll -paminobenzoyl)-glutamyl-glutamic acid gives diethyl N'-(N'-('2-keto-n-butyl) N-(p-toluenesulfonyl)"p-aminobenzoyl)'-glutamate, diethyl N- (N (2 keto-l-methyl-n-pentyl) -N-(p-toluene-' sulfonyl) -p-aminobenzoyl) glutamate, N (N- (Z-keto-n-butyl) -N-(- p-toluenesulfonyl) p-ami- 'ncbenzoyl) -glutamic acid, N- (2-ketopr0pyl)- N- (benzenesulfonyl) p aminobenzoic acid, ethyl N- (2-ketopropyl) -N-(benzenesulfony1) -p-amino'- Example, 4; Diethyl' N-(N'-(2-Iceto-3-oa:imi'1 w'- propyll-N-(p-ttilueizetulfonyl) p aminoben cowl-glutamate A mixture of 1.0 gram of diethyl N-(N-(2- lretopropyll -N-(-ptoluenesul'fonyl) -p-aminobenzoyD -glutamate; l5 milliliters of anhydroushdiethyl ether saturated with hydrogen chloride-and 0-.23' milliliter of n-butyl nitrite was stirred at room temperature for 22 hours and the volatile components then volatilized under reduced pressure. There was thus obtained 1.05 grams of diethyl .Nf-(N (2-keto-3-oximinopropy1) -N-( ptoluenesulfonyl)p+aminobenzoylglutamate as a viscous yellow oil.

Other alkyl nitrit'esfluch as ethyl nitrite, propyl nitrite and amyl-ni'trite when rea'ctedwith diethyl' N'-'(N'(2'ketopropyl )-N-(p toluenesulfonyl) -p-aminobenzoyllglutamate in the man"- ner just described lead to the formationioi" the same diethyl. .N'.- (N- (2-keto-3-oximinopro'pyl) Ngtpz-toluenesulfonyl) -p-aminobenzoyl) -glutama e..

In similar fashion. other esters} such as diethyl N ('N (2 keto nbutyl N (p toluenesulfonyl) p:- aminohenzoyl) glutamate, di ethylN j- (N' (2.- lreto a-m'e'thyl n pentyl) N (p toluenesulfony'l) p aminobenzoyl lglutamate ethyl N (2-ketopropyl) -N- (benzenesulionyl).-rr-aminobenzoate and triethyl N"-(N- (2 ketopropyl). N (p chlorobenzenesuliminopropyll-lil-(p chlorobenzenesulfonyl) -panrinobenzoyl =glutarnyl glutamate} respectively. Emm ie ii -Dignity Nf- (1v gz-tetasl-o i mo propyZl-N-lp tolu esuZ,fonyL)--paminobenzoyll olutamztte Amixture of 1.0 g'ria-m of diethyl N- (N- (2-ketopropyl) N -(p---toluenesulfonyl)' p"- amino milliliters of benzene Was stirred at room tem-- perature for 20 hours underan atmosphere of nitrogen. The benzene solution" was then extracted: with an equal volume of dilute. aqueous hydrochloric I acid, then' with an equal volume-ot water and dried,.; Upon Example 6.-N'-(N-(2 keto-3-omiminopropyl): N (p toluenesulionyl) (p aminobenzoyl) glutamic acid (N: cam-3gamma -.N (ptoluenesulfonyD -p-aminobenzoyl). -'glutamic acid 'is prepared substantially'ajs by thel'method'ojf Example 4 by treating 'N" ,-(N-(2 -ketopropyl) -N,-

(p, toluenesulfonyl) -'p-aminobenzoyl) glutamic acid with'n-butyl nitrite. in ethereal hydrogen chloride; The product is recovered by volatilizing the low-boiling constituents-of the mixture under reduced pressure.

In similar fashion other acids such as N-(N- (2 keto n butyl) N (p toluenesulfonyl) p aminobenzoyl) glutamic acid, N-(2-ketopropyl) N '(benzenesulfonyl) p aminobenzoic acid, and N (N (2 ketopropyl) N (pchlorobenzenesulfonyl) p aminobenzoyl)- glutamyl-glutamic acid are reacted with butyl nitrite to form N (N (2 keto-3-oximino-nbutyl) N (p toluenesulfonyl) p aminobenzoyl)-glutamic acid, N-(2-keto-3-oximinopropyl) N (benzenesulfonyl) p aminobenzoic acid and N'-(N-(2-keto-3-oximinopropyl)- N (p chlorobenzenesulfonyl) p aminobenzoyl)-glutamyl-glutamic acid, respectively.

Example 7.Diethyl N (N ((2 amino-el-hydroxy-fi-pteridyl) -methyZ)-N-(p tolaenesalfang Z) -p-aminobenzoyl) -glatamate A mixture of 1.05 grams of diethyl N-(N-(2- keto 3 oximinopropyl) N (p toluenesulfonyl) p aminobenzoyl) -glutamate, 428 milligrams of 2,4,5-triamino-6-hydroxypyrimidine, 328 milligrams of anhydrous sodium acetate and milliliters of glacial acetic acid was stirred at room temperature for minutes under an at mosphere of nitrogen. The mixture was then heated at its refluxing temperature and the acetic acid finally volatilized under reduced pressure. There was thus obtained a colored residue containing inorganic salts and diethyl N'-(N-((2- amino 4 hydroxy 6 pteridyl) methyl) N- (p-tolenesulfonyl) -p-aminobenzoyl) -glutamate.

In similar fashion 2,4,5-triamino-6-hydroxypyrimidine is condensed with ethyl N-(2-keto-3- oximinopropyl) N (benzenesulfonyl) paminobenzoate, N (2 keto 3 oximinopropyl) N-(benzenesulfonyl)-p-aminobenzoic acid, triethyl N (N (2 keto 3 oximinopropyl)- N (p chlorobenzenesulfonyl) p aminobenzoyl)-glutamyl-glutamate, N (N (2 keto- 3 oximinopropyl) N (p chlorobenzenesulfonyl) p aminobenzoyl) glutamyl glutamic acid, diethyl N (N (2 keto 3 oximino nbutyl) N (p toluenesulfonyl) p aminobenzoyl) glutamate and diethyl N (N (2- keto 3 oximino 4 methyl n pentyl) N- (p toluenesulfonyl) p aminobenzoyl) glutamate to form ethyl N-((2-amino-4-hydroxy-6- pteridyl) methyl) N (benzenesulfonyl) paminobenzoate, N ((2 amino 4 hydroxy 6- pteridyl) methyl) N (benzenesulfonyl) paminobenzoic acid, triethyl N (N ((2-amino- 4 hydroxy 6 pteridyl) methyl) N (pchlorobenzenesulfonyl) p aminobenzoyl) glutamyl-glutamate, N (N-((2 amino-4-hydroxy 6 pteridyl) methyl) N (p-chlorobenzenesulfonyl) p aminobenzoyl) glutamyl- :glutam'iej acid, diethyl :N'-j(N (2-amino-fl4-hydroXy-7-methy1 fi-pteridyl) methyl) N (ptoluenesulfonyl) p aminobenzoyl) glutamateand diethyl N'-(N-((2-amino-4-hydroxy-7-isopropyl 6 pteridyl) methyl) N (p toluenesulfonyl) p aminobenzoyl) glutamate, respectively.

- Example 8.-Diethyl N (z-amiao-4 hydroay 6 -,-pteriayl)fl- --;a ut tem "Th'e'crude diethyl N'-' (N amma-ma a- 6 pteridyl) methyl) N (p toluenesulfonyl) -p-aminobenzoyl) -glutamate obtained 5 in Example? and 0.4.gram of phenol were dissolved in 8 milliliters of a 30 percent solution of hydrogen bromide in glacial acetic acid. The mixture was allowed to stand at room temperature for minutes and then stirred into milliliters of dry ethyl ether. The solid which separated was recovered by centrifuging and dried. There was thus obtained a light brown residue of diethyl N (N-((2-amino-4-hydroxy-6-pterid yl) -methyl) -p-aminobenzoyl) -glutamate.

Example 9.-N'-(N-(-(2-amino 4 hydroxy 6- pterz'dyl) -methyl) -p-amz'noben2oyl) glatamic acid The dry diethyl N'-(N-((2-amino-4-hydroxyfi-pteridyl) -methyl) -p-aminobenzoyl) -glutamate as obtained in Example 8 was dissolved in a mixture of 5 milliliters of methanol and 15 milliliters of 10 per cent aqueous sodium hydroxide. The solution was allowed to stand at room temperature for one hour and aqueou hydrochloric acid was then added until the mixture was at pH 3.0.

The precipitate which formed was collected by filtering and washed with 10 milliliters of water and then with three 7-milliliter portions of acetone and finally dried. There was thus obtained milligrams of N-(N-( (2-amino-4-hydroxy-6- pteridyl) -methyl) -p aminobenzoyl) glutamic acid which, upon microbiological assay with L.

casez', had an activity of of 14.5 per cent of that of the pure natural acid.

Example 10.-N-(N-((2-amino= 4 hydroxy 6- pterz'dyl) -methyl) -N- (p-toZuene-sulfonyl) paminobenzoyD-glutamic acid Crude N- (N- (2-keto-3-oximinopropyl) -N- (pmethyl) -jamino benzoyl.)

toluenesulfonyl) -p-aminobenzoyl) -glutamic acid I as prepared in Example 6 is reacted with 2,4,5- triamino-G-hydroxypyrimidine substantially as by the method of Example 7. There is thus obtained a brownish residue containing inorganic salts and N- (N-( (2-amino-4-hydroxy-6-pteridyl) -methyl) -N- (p toluenesulfonyl) p aminobenzoyl)-glutamic acid. Treatment of the residue with phenol and a glacial acetic acid solution of hydrogen bromide substantially a by the method of Example 8 yields the same N-(N- ((2-amino-4-hydroxy-6-pteridyl) -methyl) paminobenzoyl) -glutamic acid obtained in Example 9.

We claim:

1. The method which includes: reacting a 2- keto-3-oximinoalkyl compound having the formula HON 000R H R-C-CO-CHzJITQ-OO(NHCHCH CHZCO),.OR

aryl S02 wherein R is a member of the class consisting of hydrogen and the alkyl radicals, n is a member of the class consisting of zero and the positive integer 1 and R is a member of the class consisting of hydrogen and the alkyl radicals with 2,4,57- triamino-6-hydroxypyrimidine to form a compound having the formula:

CEO-SO:

' C O 0-alky1 C ONHCHCHZCHzC O O-alkyi 12 with 2,4,5-triamino-G-hydroxypyrimidine to form a compoun'dhaving the formula 1 C O O-alkyl 11 I v- N y GHZ-NQCQNHeHCHZCmGOO-amm HEN-KN 3. The method of claim 1 wherein the 2-keto- 3-oximinoa1ky1 compound is diethy1"N-(N-(2- keto-3-oximinopropy1) -N-(p-to1uenesulf0ny1) -paminobenzoyl) -g1uta,mate.

BARNEY J. MAGERLEIN. DAVID I. WEISBLAT.

No references cited. 

1. THE METHOD WHICH INCLUDES: REACTING A 2KETO-3-OXIMINOALKYL COMPOUND HAVING THE FORMULA 